Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the...

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Bibliographic Details
Published in:Revista da Sociedade Brasileira de Medicina Tropical
Main Authors: Juliana Figueirêdo da Costa Lima, Gabriela de Moraes Rêgo Guedes, Juliana Falcão de Araújo Lima, Laís Ariane de Siqueira Lira, Fabiana Cristina Fulco Santos, Mercia Eliane de Arruda, Lílian Maria Lapa Montenegro, Haiana Charifker Schindler
Format: Article in Journal/Newspaper
Language:English
Published: Sociedade Brasileira de Medicina Tropical (SBMT) 2015
Subjects:
Mycobacterium tuberculosis
Molecular diagnostic test
Nested polymerase chain reaction
Blood
Urine
Arctic medicine. Tropical medicine
RC955-962
Arctic
Online Access:https://doi.org/10.1590/0037-8682-0210-2015
https://doaj.org/article/f3f282239a814286b26d3f3b39ad4706
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Summary:Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

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